There are more than twenty genetically distinct collagens in the body characterized by different genes, different amino acid sequences, different structures and different histological locations but with common features including being triple helical molecules with high glycine and praline content in the triple helical domains. Collagen type 1 (hereinafter “Collagen 1”) is the most abundant of the collagens and is found in large quantities in tendon, bone, skin, cornea and other sites. Collagen 1 fibers can be identified by a 67 nm periodicity, by immunoreactivity to specific antibodies and by staining with various dyes. The collagen fibril serves as one of the prominent scaffolding structures utilized in animals, where the strength of an individual ropelike collagen molecule relates directly to the structural integrity and strength of the tissue. Collagen 1 fibrils are substantial constituents of skin, tendon, bone, ligament, cornea, where the fundamental tensile properties of the fibril are finely tuned to serve bespoke biomechanical, structural, and mechanotransductory signaling roles. Many of these properties derive from the structural organization within a fibril, where the organization and topology of the collagen molecules ensure strong intermolecular interactions which are further stabilized by covalent crosslinks derived from lysines in the molecule. The presence of subfibrillar organization may point to structural levels of organization that are required for the successful mechanical response of fibrillar collagens, and may also be part of the inevitable balance between crystallinity and disorder within a biological polymer.
The principles for the self-assembly of collagen fibrils into structures resembling those found in vivo remain a mystery, though the importance of liquid crystal-like arrangements of the collagen as secreted by organized groups of cells represents a current model.
The assembly of Collagen 1 into fibril has long been regarded as a spontaneous self-assembly process, the limitation of fibril size could be ascribed to a physical equilibrium between soluble procollagen molecules and the growing insoluble fibril. Fibril-forming collagens are synthesized as precursor procollagens, where N- and C-terminal globular propeptide extensions maintain solubility. The C-propeptide directs chain association during intracellular assembly of the pro-collagen molecule from its three constituent alpha chains. During secretion and deposition as the extracellular matrix, the globular propeptides are cleaved by specific procollagen proteinases, triggering fibril formation, as illustrated in FIG. 1 as reported by M. J. Buehler, Proc Natl Acad Sci US 103, 12285-12290, 2006.
Collagen 1 is a ubiquitous protein in the animal kingdom that evolved to provide a support and framework for cells, and to give strength and resiliency to skin, bone, and tendon. Not unexpectedly, Collagen 1 has been much used as a biomaterial for many medical uses including, skin augmentation, sutures, artificial skins, dura replacements and for other uses. The science of restructuring collagen for use in biomaterials is, however, in its infancy. Much of the research up to now has been characterized by applications of different types of collagen preparations. These experiments have demonstrated that Collagen 1 can be implanted in a variety of sites in the body, and that it doesn't elicit a severe immunologic reaction. Techniques are available for solubilizing large quantities of Collagen 1 from the skin and tendons of a variety of animals or from human skin and for reconstituting the solubilized material into fibrillar form for use in humans.
Professor F. O. Schmitt and his group, working at MIT during the Second World War, investigated the use of collagen sutures and collagen sheets for covering denuded areas of skin. In a remarkable prophetic series of investigations, this group concluded that it was feasible and practical to use collagen sheets and fibers in surgical procedures. They also initiated studies on collagen tubes for nerve repair. Since then, collagen sutures have proved quite successful, with less tissue reaction and better handling properties than catgut.
Collagen 1 can be deposited from solution by a variety of process including casting, lyophilization, electrospinning and other processes well known to one skilled in the art. In most of these procedures, collagen fibers of widely varying diameters and lengths from the micrometer range typical of conventional fibers down to the nanometer range are formed, which provides a mat of interlaced fibers having interstices and pores which provide a suitable foundation for anchoring cells. Owing to their small diameters, electrospun fibers possess very high surface-to-area ratios and are expected to display morphologies and material properties very different from their conventional counterparts occurring in nature. Belamie E. et al. J. Phys. Condens. Matter, 2006, 18, 115-129.
When liquid crystals were discovered in 1888, they quickly became strong candidates for the mechanism by which nature forms living structures from homogeneous multi-chemical mixtures. This is because liquid crystals display a “striking form of self-organization in which directional order appears spontaneously in a homogeneous liquid, not incrementally, as in the growth of crystals layer by layer at the surface, but simultaneously throughout a substantial volume”. The organization of structure in a volume is slow if it only occurs through surface apposition of material as with the growth of crystals; while liquid crystals can organize and occur simultaneously over the entire volume.
Liquid crystal is a state of matter that is intermediate between the crystalline solid and the amorphous liquid. There are three basic phases of liquid crystals, known as smectic phase, nematic phase, and cholesteric phase as illustrated in FIGS. 2a-2c. FIG. 2a illustrates the smectic phase in which one-dimensional translational order, as well as orientational order exists. FIG. 2b illustrates the nematic phase in which only a long-range orientational order of the molecular axes exists. Cholesteric phase is also a nematic liquid type with molecular aggregates lie parallel to one another in each plane, but each plane is rotated by a constant angle from the next plane, as shown in FIG. 2c, FIG. 3, and FIGS. 4a-4b. The cholesteric phase is a chiral form of the nematic phase. Chiral describes a structural characteristic of a molecule that prevents it from being superposed upon its mirror image. The “twisted plywood model” shown in FIG. 3 is a model of the organization of molecules in a cholesteric structure. This model explains how typical series of arcing patterns observed in sections of cells and tissues result not from authentic curved filaments but originate from the successive molecular orientations found in the twisted plywood arrangement. The model is constructed as follows. The molecular directions are represented by parallel and equidistant straight lines on a series of rectangles, with the orientation of the lines rotating from one rectangle to the next by a small and constant angle. A periodicity is visible wherein each 180° rotation of the molecular directions corresponds to the half-cholesteric pitch P/2. The rotation is chosen to be left-handed, as has been found in all biological twisted materials studied so far. A cholesteric axis is defined by the left-hand rule, the closed fist of the left hand indicating the progressive direction of twist and the extended thumb of the left hand pointing in the positive direction of the cholesteric axis. Directly visible on the oblique sides of the pyramid are what appear to be superposed series of parallel nested arcs. The concavities of the arcs are reversed on opposite sides of the model. In biological systems this particular geometry has often been described as twisted plywood.
Two major types of twists are found in liquid crystals and their biological analogues and are defined by the disposition of the fibrillar elements either in parallel planes (planar twist) or coaxial cylinders (cylindrical twist) (see FIG. 4). The coaxial cylinders or cylindrical twist are also described as helicoidal. Collagen in the secondary osteons of bone tissue is observed to be in a helicoidal pattern as is cellulose in plant cell walls.
Additional techniques are needed for forming composite collagen-based films. One technique recently reported used an inkjet printer capable of printing at high resolution by ejecting extremely small ink drops. Researchers hope that established printing technology will be able to seed living cells, at micrometer resolution, in arrangements similar to biological tissues. This method was described in Nakamura M. et al., Tissue Engineering, 11:1658-1666 (2005) wherein the authors used a biocompatible inkjet head and investigated a feasibility of microseeding with living cells. Living cells are easily damaged by heat; therefore, they used an electrostatically driven inkjet system that was able to eject ink without generating significant heat. Bovine vascular endothelial cells were prepared and suspended in culture medium, and the cell suspension was used as “ink” and ejected onto culture disks. Microscopic observation showed that the endothelial cells were situated in the ejected dots in the medium, and that the number of cells in each dot was dependent on the concentration of the cell suspension and ejection frequency chosen. After the ejected cells were incubated for a few hours, they adhered to the culture disks. While these developments have been made, this technique is limited and has not found widespread use. This technique is somewhat useful for delivering a material (e.g., cells) to a particular area but it cannot maintain and preserve the material's orientation.
All prior art methods of forming collagen films and matrices to date suffer from limitations as do the collagen-based materials formed there from. The main limitation is to maintain and preserve the native liquid crystal structure of collagen-like materials. For example, electrospinning and casting methods cannot preserve a long-range orientation. Collagen based films and matrices cannot mimic the native semi-crystalline structures of the extra-cellular matrix in the living biological systems. Other methods, like, for example, Langmuir-Blodgett method, have limited orientation and poor repeatability. Accordingly, there is significant need for new collagen-based materials mimicking native structures in living biological systems as well as the reliable and robust methods of producing such materials.